versión On-line ISSN 0717-9502
Int. J. Morphol. v.26 n.2 Temuco jun. 2008
Int. J. Morphol., 26(2):377-384, 2008.
Research of Torque teno virus (TTV) in Serum and Total Blood of Brazilian Non-Human Primates and in Chicken Plasma (Gallus gallus domesticus) by the PCR N22 Region
Investigación de Torque teno virus (TTV) en Suero y Sangre de Primates No Humanos Brasileños y en Plasma de Pollo (Gallus gallus domesticus) por PCR en la Region N22
*,**Catroxo, M. H. B.; ***Nishiya, A.; ***Sabino, E.; ****Teixeira, P. S.; *****Petrella, S.;******Milanelo, L.; *******Vieira, J.C.F. & **Diaz, R. S.
*Laboratory of Electron Microscopy, Institute Biological of São Paulo, Brazil.
SUMMARY: Torque teno virus (TTV) is a recently discovered DNA virus that was originally isolated from a Japanese patient (initials, TT) with post-transfusion hepatitis of unknown aetiology. TTV is an circular DNA virus classified recently together with related Torque teno minivirus, into a new genus called Anellovirus. Infection TTV has been detected in a range of non-human primates as well as domestic animals. The purpose of this study was to search TTV in the serum and total blood of Brazilian monkeys and in plasma of domestic chickens by seminested PCR of coding region (N22), followed by a genomic sequence and phylogenetic analysis. No serum sample was amplified. TTV DNA was detected in total blood from 3 (4%) out of 75 brown-capuchin (Cebus apella) and from 1 (25%) out of 4 golden-headed lion-tamarin (Leontopithecus chrysomelas). Phylogenetic analysis revealed that one sample showed similarity with one sequence of the cotton top tamarin (Saguinus oedipus) (So-TTV2) and with one of the douroucoulis (ão tes trivirgatus) (At-TTV3). Two samples showed similarity with a human Torque teno Mini Virus (TLMV). The other sample clustered with one sequence of the chimpanzee (Pt-TTV6) and with the human TTV strain TA278. The plasma chicken samples tested were all negative. The amino acid sequences reported in this study are the first obtained in Brazil from total blood of non-human primates naturally infected by TTV.
KEY WORDS: Torque teno virus; Non-humans primates; Coding region; Amino acid sequencing.
RESUMEN: Torque teno virus (TTV) es un virus de ADN recientemente descubierto que fue inicialmente aislado de un paciente japonés (iniciales TT) después de la transfusión de hepatitis de etiología desconocida. TTV es un virus de ADN circular recientemente clasificado junto con los torque teno minivirus, en un nuevo género llamado Anellovirus. La infección de TTV se ha detectado en una serie de primates no humanos, así como animales domésticos. El objetivo de este estudio fue buscar TTV en el suero y sangre total de monos de Brasil y en el plasma de pollos domésticos, por seminested PCR de la región de codificación (N22), seguido de una secuencia genómica y el análisis filogenético. Las muestras que no eran suero fueron amplificadas. TTV DNA se detectó en sangre total de 3 (4%) de un total de 75 capuchinos de cabeza dura (Cebus apella) y de 1 (25%) de un total de 4 tití- león de cabeza dorada (Leontopithecus chrysomelas). El análisis filogenético demostró que una muestra presentaba similitud con una secuencia de Saguinus Edipo (So-TTV2) y con una de Aotes trivirgatus (A-TTV3). Dos muestras mostraron similitud con un torque teno mini virus (TLMV) humano. La otra muestra agrupada con una secuencia de los chimpancés (PT-TTV6) y con el TTV humanos cepa TA278. El análisis de las muestras de plasma de pollo fueron negativas Las secuencias de aminoácidos que se reportan en este estudio son las primeras obtenidas en Brasil de sangre de primates no humanos infectados naturalmente por TTV.
PALABRAS CLAVE: Torque teno virus; Primates no humanos; Región de codificación; Secuencia de aminoácidos.
Torque teno virus (TTV) is a recently discovered DNA virus that was originally isolated from a Japanese patient (initials, TT) with post-transfusion hepatitis of unknown aetiology (Nishizawa et al., 1997). Since discovery of TTV, studies have been published describing the prevalence of TTV infection in people with acute or chronic hepatitis as well as in blood in health persons (Biagini, 1998; Mac Donald et al, 1999).donors and drug users and also TTV is an unenveloped, single-stranded, and circular DNA virus with 30-32 nm of diameter (Miyata et al, 1999; Mushahwar et al, 1999; Itoh et al, 2000), classified recently together with the related Torque teno Minivirus, into a new genus called Anellovirus (Biagini et al, 2005).
TTV is transmitted parenterally by transfusion (Okamoto et al, 1999), byenteric transmission demonstratedexperimentally in chimpanzees (Luo & Zhang, 2001; Tawaraet al, 2000), and by vertical transmission (Saback etal, 1999). Also has been detected in saliva, on throat swabs, semen, tears, breast milk, hair, skin and raw (Saback et al; Goto et al.,2000;Inami et al.,2000;Osiowy & Sauder,2000; Al-Moslih et al, 2007). Infection TTV has been detected in a range of non-human primates (Leary et al, 1999; Verschoor et al, 1999; Abe et al, 2000; Cong et al, 2000; Okamoto et al, 2000a,b; Noppornpanth et al, 2001; Thom et al, 2003; Barnet et al, 2004; Pujol et al, 2005) as well as domestic animals, such as dogs, cats, swines, bovines, chickens, ovines, tupaias (thee shrews), wild boar (Sus scrofa) and camels (Leary et al.; Okamoto et al, 2001a; 2002; Me Keown et al, 2004; Bigarré et al, 2005; Niel et al, 2005; Martelli et al, 2006; Martínez et al, 2006; Al-Moslih et al).
Study of experimental transmission points out that the chimpanzees are susceptible to TTV infection and that can be cross infected with human (Mushahwar et al; Okamoto et al, 2000a, b).
TTV genome includes two regions: a coding region (N22) and an untranslated region (UTR). The UTR is located atnt3075-3853 andnt 1-352 occupying approximately 30% of the genome (Okamoto et al, 1999; Ukita et al, 2000). Coding region consist of 6 ORFs with open reading frames (ORF1-ORF6) (Yokoyama et al, 2002). The N22 amplifies a fragment in open reading frame 1 (ORF1) and represents approximately 220 nts of a genome that usually encompasses more than 3800 nts (Miyata et al; Mushahwar et al). Consequently, almost every phylogenetic analysis of TTV is focused on this genomic region (Lemey et al, 2002).
The significant diversity of the TTV genome allows for its classiñcation into more than 40 genotypes which clus-ter in five clearly distinct phylogenetic groups that are designated 1 to 5 (Okamoto & Mayumi, 2001; Hino, 2002; Peng et al, 2002; Biagini et al).
Other members of this virus family (SANBAM, YONBAN, TLMV (Torque teno Minivirus), SENV (SEN virus) and PMV (PM virus), presenting some genomic divergences have been related (Cong et al; Hallet et al, 2000; Khudyakov et al., 2000; Takahashi et al, 2000b; Diniz-Mendes et al, 2004).
The purpose of this study was to search TTV in the serum and blood of Brazilian monkeys and plasma of domestic chickens (Gallus gallus domestícus) by PCR of coding region (N22), followed by a genomic sequence and phylogenetic analysis.
MATERIAL AND METHOD
Animals: Were collected from August, 2000 to February, 2001, 90 serum samples and 70 total blood samples from various non-human primates, both sexes, including 26 Callithrix jacchus, 3 Callithrix kulii, 4 Leontopitecus crysomelas, 75 Cebus apella, 19 Calllithrix penicilata, 2 Callithrix sp, 5 Alouata carayá, 5 Alouata fusca, 1 Saguinus m. niger, 1 Callithrix humeralifer, 3 Callithrix geoffroyi, 1 Aotus trivirgatus, 1 Calimico goeldi, 3 Saguinus m. midas, 1 Lagothrix lagotricha, 1 Erytrocebus pata, 2 Ateles b. marginatus, 1 Ateles penis cus, 4 Saimiri sciureus, 1 Saimiri fuscicollis, 1 Leontopitecus chrysopygus. These species are usually found in the Amazon Forest and Atlantic Rainforest (Mata Atlántica), in Brazil. These animals carne from donations or rescues and were apprehended and sent to several ecological andZoological Parks in São Paulo, Brazil where they were maintained in captivity. A total of 117 samples of chicken plasma (Gallus gallus domestícus) of the following lineages: white, brown leghorn and hens of yard, of both sexes from different poultry farms in the State of São Paulo, SP, Brazil, were collected from May to September 2001. Samples were kept at -80°C until testing.
Extraction of nucleic acids and amplification by PCR: Blood DNA was extracted using a DNA Extraction Kit (Qiagen) from 100 µl total blood. DNA was resuspended in 100 µl buffer. Serum DNA (50 µl ) was mixed with 100 µl Dnazol and 3 µl Dextran T500 (1mg/µl ). The DNA was precipitated with isopropanol and resuspended in 25 ul distilled water.
Heminested N22 PCR: In the first round, outer primers NG059 and NG063 (sequences 5' ACA GAC AGA GGA GGG AAC ATC - 3' and 5' - CTG GCA TTT TAG CAT TTC CAA AGTT - 3') were used.
PCR was carried out in 50 µl volume, using 5 µl of DNA, 10 pmol of NG059 and NG063 primers, 1.5 mM MgC12 and 5U Taq polymerase. In the second round, semi-inner primer NG061 (sequence 5' - GGC AAG ATG YTR TGG ATA GAC TGG - 3') and the outer primer NG063 were used. Five µl of the first PCR product and 10 pmoles/ µl of NG061 and NG063 primers were used for the second PCR. Ampliñcations conditions were 94°C 30s, 55°C 30s, 72°C 30s, 35 eyeles, with a final extension at 72°C 7 min.
The amplification products of the first PCR round were 286 pb, and those of the second round were 271bp. It was considered positive for the TTV control, human serum from a TTV positive patient, and negative, ultra pure sterile water, free from inhibitors or contaminants, which could avoid amplification of PCR products. Amplified products were separated by electrophoresis through a 2% agarose gel containing ethidium bromide, and visualized under UV light.
Sequencing. Sequence reactions were performed with the ABI Prism Big Dye Terminator Cycle sequencing Ready Reaction Kit (Applied Biosystems). Cycle sequencing was carried out with an automatic DNA Sequencer (ABI Prism 377 Sequencer - Applied Biosystem). The primers used were NG061 and NG063 of the N22 region. Sequence edition was performed using the Sequencher program 4.0.5 (Gene Code Corporation). Sequence alignments were generated by the Bioedit Program (Hall, 1999). The phylogenetic analysis was performed using Philip version 3.5 c (Felsenstein, 1993).
Alignment with 3 edited sequences from the N22 region and series of standard sequences from different TTV genotypes from Gen Bank and from the literature were performed.
The sequence alignments were assessed using 1000 bootstrap replicates using SEQBOOT Program (Felsentein). Phylogenetic trees were constructed by the neighbor-joining method (Saitou & Nei, 1997). The consense tree was calculed using the Consense Program and visualized in the Treview Program version 1.6.6 (win 32) (Roderic, 2001). The genotypes of the TTV had been determined by comparing the lined sequences (GenBank accession numbers EU919749-EU919752).
Non-human primates. Of 160 samples obtained from different species of new world non-human primates (90 serum and 70 whole blood) analyzed by the semi-nested PCR reaction for the N22 region only 4 samples of total blood were amplified, generating a band with a 271 pb fragment in the second round. No serum sample was amplified.
TTV N22 DNA was detected in total blood from 3 (4%) of 75 brown capuchin (Cebus apella) and from 1 (25%) of 4 golden-headed-lion-tamarin (Leontopithecus chrysomelas). Among these 2 (50%) were males and 2 (50%) female, all adults (Table 1).
Phylogenetic analysis revealed that one sample showed similarity with one sequence of the cotton top tamarin (Saguinus oedipus) (So-TTV2) and with one of the douroucoulis (Aotes trivirgatus) (At-TTV3). Two samples showed similarity with a human Torque Teno Mini Virus (TLMV). The other sample clustered with one sequence of the chimpanzee (Pt-TTV6) and with the human TTV strain TA278 (Fig. 2).
Gallus gallus domesticus. The 117 plasm samples tested by the semi-nested PCR reaction for the N22 region were all negative.
The diagnostic of TTV infection is frequently realized by DNA amplification by PCR utilizing primers designed or by coding region of the ORF 1 (N22) or by non-coding region (UTR) of the viral genome (Ott et al, 2000).
The phylogenetic analysis of the TTV ha ve been based on a small genome region that exhibits extensive sequence heterogeneity (Lemey et al).
Our results obtained by the semi-nested PCR reaction for the N22 region revealed amplification in 4 (5.7%) out of the 70 samples of total blood of new world non-human primates. However, no amplification was observed in 117 serum of the animals submitted to the same reaction.
On the contrary, other studies did not obtain positivity by the PCR of the N22 region, in samples of non-human primates of the old and new world (Abe et al; Pujol et al), not even in pig, cat and dog samples (Okamoto et al, 2002).
Studies in the literature have demonstrated variations in positivity percentage in samples of the old world non-human primates. Nopporpanth et al. detected 9 (13.4%) of 67 gibbons, Thom et al. 1 (20%) of 5 chimpanzees, Romeo et al. (2000) 2 (25%) out of 8 naive chimpanzees, Verschoor et al. 60 (48.%) out of 123 chimpanzees e 4 (66.%) out of 6 pygmy chimpanzees, Barnett et al., 4 (80%) of 5 chimpanzees of the Congo community and 3 (100%) of 3 chimpanzees of the Sonso community of the Budongo Forest Reserve of Uganda.
Using specific primers for each species of chimpanzee, Okamoto et al. (2000a) detected 57% in 49 out of 104 chimpanzees, but they did not obtain positivity in any sample that they have tested with new world non-human primates. No other study in the literature has detected the presence of DNA TTV in new world non-human primates by the N22-PCR.
Okamoto et al. (2000a) e Leary et al., however, detected DNA TTV in new world monkeys by PCR of the non-coding region (UTR), since the primers used in the N22-PCR only allow the amplification of a restricted number of TTV genotypes (Bendinelli et al, 2001; Vasconcelos et al, 2002).
In this study DNA TTV was detected in 3 samples of Cebus apella and in 1 sample of Leonthopithecus crysomelas. Abe et al. however, had detect DNA TTV in none of the 9 Cebus apella samples submitted to N22-PCR .
Cebus apella is the greatest specie, largely distributed in South América, present in all Amazon Forest, swamp, woodsy pasture, upland caatinga and Atlantic Rain Forest (Silva Junior, 2002).
In our study one sequence of the Cebus apella clustered with one strain isolated from Saguinus oedipus (So-TTV2) and other with from ão tes trivirgatus (At-TTV3) of the Okamoto et al. (2000b).
Okamoto et al. (2000b) performed by cloned from UTR-PCR products representing the full-length TTV genomes obtained from sera of humans and non-human primates that showed common genomic organization with two open reading frame (ORFs), designated ORFl and ORF2 and their sequences were determined. In accordance with the author, these sequences bifurcated from the same branch harboring Japanese macaque TTV, reflecting a close relationship of these lower classes non-human primates TTVs.
Our results showed that one sequence clustered with a human TLMV (Torque Teno Mini Virus) sequence.
Furthemore, TTV and TLMV DNA sequences have been found in non-human primates and farm animals (Verschoor et al, 1999; Abe et al, 2000; Leary et al, 1999; Okamoto et al, 2000 a, b; Romeo et al., 2000).
TLMV is a unique virus with a smaller genome that may constitute a genetic evolutionary link between chicken anemia virus and TTV (Takahashi et al, 2000a).
In this study, 1 amplified sample (Cebus apella) was grouped with one strain of chimpanzee (Pt-TTV6) (Okamoto et al, 2000b) and with the TA278 human prototype strain.
A close proximity was observed, between the gibbon virus and those detected in Thai individuals , whereas that, chimpanzee strains were phylogenetically more remote (Noppornpanth et al).
According to Verschoor et al. TTV from pygmy chimpanzees and the common chimpanzees closely to virases from human TTV from the common chimpanzees subspecies Pan troglodytes verus and Pan troglodytes schweinfurthü cluster together, suggesting an ancient host-pathogen relationship before sub speciation 1.6 million years ago and TTV of common and pygmy chimpanzees may have been acquired by these animals in different zoonotic events not longer than 2.5 million years ago.
Abe et al. reported one study that all TTV isolates obtained from simians (s-TTV) (old world non-human primates) were clearly distinct from TTV found in humans.
Inami et al. stated that a strain CH65-1 in ORF-1 region showed only 35% identity to the prototype TA278 human isolate at the amino acid level.
The results of our research showed that the sequence of DNA TTV obtained of the Cebus apella (Ca-TTV17) were close to human TTV of the genotypel.
In the same manner, gibbons sequences (Nopporpanth et al) and in pygmy chimpanzees Okamoto et al. (2000a, b) were grouped in genotype 1.
Verschoor et al. however, reported that TTV sequences from pigmy chimpanzees are closely related to virases from human genotypes 2 and 3, while that sequences obtained from common chimpanzees were genotypes 5 and 6, the latter only at the protein level.
The phylogenetic analyses of the ORFl nucleotide consensus sequences suggested that TTV recovered from two chimpanzees (CH1304 and CH 1545) represented two new genotypes (Romeo et al).
Simian TTV (s-TTV) was further divided into three genotypes and showed type 1 as the major genotype (Abe et al). In this study, it was not possible to detect DNA TTV by N22-PCR in 117 samples chicken plasma.
Only two reports refer to TTV research in chicken by PCR of the non-coding region (UTR). Thom et al. had investigated the presence of virus in 29 chicken samples, as well as the 20 samples from cows, 20 from sheep and 20 from goats, but all were PCR-Utron egative. Also, Leary et al. detected the virus in various farm animal species (chickens, pigs, cows and sheep) and verified that 19% of chickens were positive by PCR-UTR, whose sequences clustered with human TTV. This different result are due to the fact of using primers deduced from a coding region sequence (N22).
The primers of the N22 region only allow the amplification of a restricted number of TTV genotypes (Bendinelli et al; Vasconcelos et al). Our results indicate that in spite of the TTV has been detected worldwide in wide scale in various animal species, in Brazil, however, their presence in new world non-human primates, in captivity and in chickens occurs in low number.
All the animals were clinically healthy, but very little is known, however, about the TTV infection of new world primates. The virus might have been transmitted from human handler to animals in the process of feeding or the wise han-dling the monkeys.
According Luo & Zhang TTV is transmitted both by blood and enteric routs and perhaps in áreas with inadequate hygienic standards, the virus is predominantly spread via the fecal-oral route.
The amino acid sequences reported in this study are the first obtained in Brazil from total blood of non-human primates naturally infected by TTV. The amino acid identity between human and non-human primates sequences in genotype 1 observed in this study supports the evidence that TTV is a zoonotic agent.
Further studies to evaluate the pathology, genetic diversity and host range, transmission, and persistence of TTV infection, in human and animal species will be conduced in order to outline considerable conclusions.
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Prof. Dr. Márcia Catroxo
Received: 24-01-2008 Accepted: 14-03-2008